HIV-1 Nef is an important virulence factor that down-regulates CD4 and class I MHC from the cell surface and enhances viral infectivity and replication. The central hypothesis of this proposal is that Nef modulates the cellular membrane trafficking machinery to induce these effects. This modulation occurs at the cytoplasmic face of membranes, where various cellular proteins control vesicle formation, the sorting of specific transmembrane proteins, and the destination of the transport vesicles within the cell. The goal here is to determine how Nef manipulates or subverts these processes. Nef interacts via an ExxxLL motif with a subset of vesicle coat-components termed adaptor protein (AP) complexes, but the roles of the different complexes in Nef-function are poorly understood. These roles will be identified using three approaches: 1) systematic mutation of the ExxxLL motif; 2) depletion of specific subunits of the complexes using siRNAs; and 3) expression of mutant subunits that block the ability of Nef to usurp the complex. Nef also dysregulates the association of AP complexes with membranes, apparently bypassing the normal attachment mechanism, which relies on the cellular protein ARF1. This activity correlates with Nef-function. The mechanism of this effect will be investigated by determining whether Nef is sufficient for the attachment process and the extent to which Nef is mechanistically similar to activated ARF1. Such a similarity could be based on direct binding to the AP complexes, the modification of membrane phospholipids, or function via a common effector protein. At least one Nef function appears to require membrane systems regulated by another ARF-family member, ARF6; this relationship will also be characterized. The modulation of AP complexes by Nef is related genetically to its virologic effects: the ExxxLL motif in Nef is required for optimal viral infectivity. A substantial portion of this effect is independent both of CD4 and of the incorporation of Env into virions. Data herein indicate that mutation of anAP binding motif in gp41 yields a phenotype similar to that of Nef-negative virus: an Env-independent defect in viral entry. We will test the hypothesis that the sorting motifs in both Nef and gp41 are involved in regulated exocytosis, targeting morphogenesis to microdomains of the plasma membrane that render the virion more fusogenic.